Immune privileged and modulatory progenitor cells

ABSTRACT

Described herein is a method for modulating an immune reaction between lymphocytes and a body recognized by the lymphocytes as foreign. The method exploits the immunomodulating activity of a new class of progenitor cells termed HUCPVCs derived from the perivascular region of human umbilical cord. The method can also emply soluble factors exuded by cultured HUCPVCs. The method is useful to treat immune disorders including graft versus host disease, autoimmune disorders, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/299,736 (now U.S. Pat. No. 8,277,794), having a 371 (c) date of Jul.8, 2009, which is the U.S. National Stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/CA2007/000781, filed May 4, 2007,which claims benefit of U.S. Provisional Patent Application No.60/746,500, filed May 5, 2006.

FIELD OF THE INVENTION

This invention relates to progenitor cells that are immunoprivilegedand/or immunomodulatory, their production, their formulation, and theirtherapeutic use.

BACKGROUND TO THE INVENTION

Adult bone marrow (BM) is the most common source of mesenchymalstem/progenitor cells (MSCs), (also called Mesenchymal Stromal Cells¹)which are functionally defined by their capability of differentiatinginto the skeletal tissues: bone²⁻⁴, cartilage⁵⁻⁷, fat⁸ and muscle⁹ invitro. MSCs are classically distinguished from the heterogeneous milieuof cells through adhesion to tissue culture plastic and the formation ofcolony unit-fibroblasts (CFU-Fs), the frequency of which are1:100,000-1:500,000 nucleated cells in adult marrow¹⁰, and studies havenow identified a suite of markers with which MSCs arecategorized^(10,11). This low proportion of MSCs leads to the necessityof culture expansion and selection before use to attain the appropriatecell numbers for any kind of cellular therapy. There are other emergingsources of MSCs such as: adipose tissue¹², trabecular bone¹³ and fetalliver¹⁴ which have a CFU-F frequency of: 1:32¹⁵, 1:636¹³ and 1:88,495¹⁴respectively. While adipose tissue does appear to have the highestfrequency of progenitors, the doubling time of those cells rangesbetween 3.6 to 4.4 days¹⁵, and the extraction procedure is complicated,invasive, and lengthy¹². Harvesting trabecular bone results in low cellyield (89×10⁶ cells/gram of bone from young donors¹³), especially whencombined with the CFU-F frequency; and is extremely invasive resultingin donor site morbidity.

Unique among these new sources of MSCs are human umbilical cordperivascular cells (HUCPVCs), which are an easily accessible, highlyproliferative source of cells with a population doubling time of 20hours (dependent on serum)¹⁶. The frequency of CFU-Fs in HUCPVCs is1:300 at passage 0 but increases to 1:3 at passage 1¹⁷, which is ordersof magnitude higher than bone marrow¹⁶. Therefore HUCPVCs represent apopulation of cells with an extremely high proportion of MSCs whichproceed to divide very quickly, thus making them an excellent candidatefor clinical mesenchymal therapies. These cells have been used invarious assays to determine their marker expression phenotype anddifferentiation potential^(16,18), and have been found to be eitherbioequivalent to, or perform better than, BM-MSCs.

In addition to their ability to differentiate, MSCs also have potentialimmunological uses as BM-MSCs have been shown to be bothimmunoprivileged and immunomodulatory¹⁹⁻²¹. These terms refer to acell's ability to evade recognition from a mismatched host's immunesystem, and the ability to mitigate an ongoing response by that system,respectively. MSCs from several sources other than bone marrow have beentested for their immunogenicity in in vitro cultures. MSCs from adiposetissue derived from adult dermolipectomies were shown to be capable ofboth immunoprivilege and immunomodulation in vitro²², whereas fetalliver cells were found to be capable of avoiding a mismatched immuneresponse, however they were not able to modulate alloreactivity causedby two mismatched populations of lymphocytes^(23,24). Thus, the sourceof MSCs directly affects those cells' immunogenic capabilities.

This in vitro work has begun to be validated in the clinical setting;for example, a boy was rescued from severe acute graft vs. host disease(GvHD) by transfusion of haploidentical bone marrow MSCs from hismother²⁵. One year post treatment, in comparison to a cohort of patientssuffering from the same level of severity of the disease, he was theonly one alive. Since this initial patient, a suite of 8 patients havebeen treated with BM-MSCs, of which 6 showed a complete remission ofsymptoms²⁶. Allogeneic BM-MSCs have also been used in Crohn's Disease totreat patients who are refractory to current treatments, and thistreatment is currently in clinical trials in the United States^(27,28).Fetal liver MSCs have shown efficacy in the early treatment ofosteogenesis imperfecta (OI). MSCs from a male fetal liver weretransplanted into an unrelated 32 week female fetus with severe OI, whohad suffered several intrauterine fractures²⁹. Following thetransplantation, the remainder of the pregnancy proceeded normally, andthere were no further fractures. This patient has been followed up to 2years after birth, and the child has shown a normal growth curve and hassuffered only 3 fractures. Using an XY-specific probe, the patient wasfound to have 0.3% engraftment in a bone biopsy.

In addition to undifferentiated cells, osteogenically induced rabbitBM-MSCs were found to be immunoprivileged and immunomodulatory in vitro,but when transplanted in vivo the immunomodulatory capacity was lost³⁰.This would not affect the function of the cells however; as they onlyrequire protection from an immune response in order to fulfill theirrole. In a more involved induction, murine bone marrow MSCs weremanipulated to release erythropoietin and implanted in mice, whichresulted in significantly less engraftment compared to un-manipulatedcontrols³¹. Thus, manipulation of MSCs can lead to their loss ofimmunomodulation and/or immunoprivilege and can be crucial to thesurvival and function of the graft.

There is evidence to support that the immunoprivilege of MSCs transcendsspecies barriers, and they can be used xenogeneically. This was firstdemonstrated by Bartholomew et al who used human BM-MSCs in baboons, andshowed enhanced skin graft survival²¹. While the end result of thisstudy was positive, the specific fate of the administered cells was notdetermined. Wang et al. have utilized GFP transfected cells andhistological analyses to studied the survival of xenogeneic BM-MSCs, andshowed that the cells survive up to the 11 week timepoint withoutimmunosuppression, however there was an increased host immunereaction³². MSCs have also been reported to survive in xenogeneictransplantations in two cardiac models^(33,34). In preliminary work withHUCPVCs, the cells were delivered peritoneally in permeable chambers.After 3 weeks, there was no noticeable inflammation noted uponmacroscopic visualization³⁵. This is encouraging preliminary workindicating the potential for not only the immunoprivilege of HUCPVCs,but also for their ability to test them in animal models withoutrejection.

The inventors investigated the immunoprivileged and immunomodulatoryproperties of HUCPVCs in vitro by conducting both: co-cultures ofHUCPVCs with unmatched lymphocytes, and mixed lymphocyte cultures (MLCs)populated by two HLA mismatched donors. Also studied were HUCPVC death,lymphocyte proliferation and activation with varying levels of HUCPVCspresent in both naïve and activated lymphocyte environments. Inaddition, the necessity for cell contact for the observation ofimmunological effects was investigated.

SUMMARY OF THE INVENTION

The inventors now report herein a series of experiments which illustrateboth the immunoprivileged and immunomodulatory capabilities of HUCPVCswhen tested in one and two-way in vitro mixed lymphocyte cultures(MLCs). Additionally, MLCs were performed which reveal a HUCPVC-induceddecrease in activation of previously stimulated lymphocytes. Theinventors further show that the HUCPVC immunomodulatory function ismediated through a soluble factor(s) produced upon culturing of theHUCPVCs, as cell contact is not required for the immunomodulatory effectto be observed. Furthermore, the inventors illustrate that HUCPVCs arecapable of modulating a two-way in vitro MLC, and describe the use ofthese cells for cellular therapy applications, particularly to modulatethe immune response.

Thus, in one of its aspects, the present invention provides a method fortreating a subject having, or at risk of developing, an adverse immunereaction, comprising the step of administering to the subject animmunomodulating effective amount of (1) a cell population comprising,and preferably consisting essentially of, human umbilical cordperivascular cells (HUCPVCs), and/or (2) an immunomodulating solublefactor produced upon culturing of said cells. In related embodiments,the method is applied to treat recipients of allogeneic or xenogeneicgrafts, including cells, tissues and organs, to reduce the onset orseverity of adverse immune reaction thereto, including graft versus hostdisease. In a general aspect, the present invention thus provides amethod for modulating an immune reaction between lymphocytes, such asperipheral blood lymphocytes, and a body recognized by the lymphocytesas foreign, comprising the step of introducing a formulation comprisinga physiologically tolerable vehicle and HUCPV cells or immunomodulatingsoluble factors that are extractable therefrom, in an amount effectiveto modulate and particularly to inhibit or reduce that immune reaction.

In a related aspect, the present invention provides for the use ofHUCPVC cells or an immunomodulating soluble factor produced thereby inthe manufacture of a medicament for the treatment of a subject having orat risk of developing an adverse immune reaction, or for the treatmentof a graft prior to transplantation, to mitigate or reduce immunereaction between the graft and recipient.

In another of its aspects, the present invention provides a formulation,in unit dosage form or in multidosage form, comprising animmunomodulating effective amount of HUCPVCs and/or an immunomodulatingsoluble factor produced thereby, and a physiologically tolerable vehicletherefor.

In a further aspect, the present invention provides an immunomodulatingextract, or an immunomodulating fraction thereof, comprising one or moresoluble factors produced by cultured HUCPVCs.

In another aspect, the present invention provides a treatment method asdescribed hereinabove, wherein the administered cells are obtained andadministered without cryogenic storage.

In a further aspect of the present invention, the administered HUCPVCsare immunoprivileged and immunomodulatory cells. In embodiments, theHUCPVCs are substantially lacking both the MHC class I and MHC class IIphenotypes. In a related embodiment, the administered HUCPVCs areobtained by thawing of a population of frozen HUCPVCs.

In a further embodiment of the present invention, the administeredimmunoprivileged HUCPVCs are engineered genetically, and incorporate atransgene that encodes a heterologous protein of interest, particularlybut not exclusively including a protein effective to manage the immunesystem such as a protein that enhances immunomodulation, and especiallya protein that inhibits adverse immune reaction, such as CTLA4.

These and other aspects of the present invention are described ingreater detail below, with reference to the accompanying Figures, inwhich:

BRIEF REFERENCE TO THE FIGURES

FIG. 1: Cell counts of HUCPVCs after 7 days in culture post-MMCtreatment (n=2). The cells were treated with ranging concentrations ofMMC for 20 minutes at 37° C. at 5% CO₂ and assayed for theirproliferation. All are seen to be significantly lower than control(p<0.001).

FIG. 2: Proliferation of cells plated in wells treated and untreatedwith MMC. Proliferation was measured using flow cytometry for BrdU, andquantified using mean fluorescence intensity. There was no significantdifference between treated and untreated wells (p=0.62).

FIG. 3: HUCPVC death was measured using mean fluorescence intensity(MFI) for annexin 5, an early cell death marker, after 4 hours ofco-incubation with PBLs from Donor 1 (n=5). There was a significantincrease in average annexin 5 expression in the culture with 10% HUCPVCsrelative to control (p=0.01, indicated by *); this was the onlysignificant increase.

FIG. 4: Lymphocyte proliferation was measured using mean fluorescenceintensity (MFI) for BrdU, after 6 days of co-incubation with varyinglevels of HUCPVCs (n=5). There was a significant increase in averageBrdU expression in the culture with 10% HUCPVCs relative to control(p=0.02, indicated by *).

FIG. 5: Total lymphocyte cell number was measured from day 1 to 6 acrosstreatments of 10 and 40% HUCPVCs added on day 0, 3 or 5 (n=3). It can beseen that by day 6, the control lymphocytes have increased in number inresponse to each other, while the treatments with HUCPVCs weresignificantly lower regardless of percentage or day added (p<0.05,indicated by *).

FIG. 6: Total lymphocyte count was measured from day 1 to 6 acrosstreatments of 10 and 40% HUCPVCs added on a TransWell™ insert (n=3). Itcan be seen that on any day, there is no significant difference betweenthe allogeneic control and the HUCPVC treatments.

FIG. 7: HUCPVCs do not increase resting or activated lymphocyte cellnumber. Addition of HUCPVCs showed no significant increase in lymphocytecell number compared to controls over 6 days in culture (n=6). ThisFigure shows the average cell numbers, + standard deviations.

FIG. 8: BrdU expression of PBLs in a co-culture with HUCPVCs measuredwith flow cytometry. The percentage of cells dividing does not increasewith addition of HUCPVCs, irrespective of dose (n=3). Control was theBrdU expression of the PBLs without HUCPVCs.

FIG. 9: HUCPVCs act through a soluble factor. HUCPVCs are able tosignificantly reduce lymphocyte cell number in MLCs, when separatedusing a TransWell insert. The average control lymphocyte cell number hasbeen set to 100% in the Figure to reduce the variation in counts betweenexperiments. This figure shows average percentage lymphocyte cellcounts, + standard deviation (n=6). (p*<0.05)

FIG. 10: HUCPVCs reduce CD25 expression in co-cultures of activatedlymphocytes. This Figure illustrates both the average percentageexpression (bar) and mean fluorescence intensity (line) of CD25expression on lymphocytes co-cultured with and without 10% HUCPVCs.Lymphocytes were stained with PKH26 to ensure proper detection of thepopulation, and results are gated on PKH26 expression. Averages are +standard deviations (n=3). (*p<0.05)

FIG. 11: CD45 expression in a two-way MLC (with activated lymphocytes)with HUCPVCs. The activated lymphocytes were stained with PKH26 todelineate them from the inactive population, and results are gated onPKH26 expression. Both percentage expression (bar*p<0.05) and MFI(line+p<0.05) are shown (n=3). Control was the CD45 expression of theATL with no HUCPVCs.

FIG. 12: (A) HUCPVCs transfected with Green Fluorescent Protein (GFP),with an expression level of 97.89% (B). These cells were transfectedwith a lentiviral vector, using established techniques, and

FIG. 13: High-throughput Cancer Pathfinder Gene Array results for bonemarrow-derived MSCs (A) and HUCPVCs (B). Genes in parentheses representthose genes which are absent.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

The present invention provides novel and clinically useful applicationsof HUCPVCs, particularly in the treatment of conditions that wouldbenefit from a reduction in the adverse response elicited byalloantigenic and xenoantigenic bodies, resulting either from an adverseimmune response by the host, or from an adverse immune reaction by theantigenic body to the host. More generally, the present inventionprovides a method in which HUCPVCs and/or soluble factors produced bythem are introduced to inhibit or reduce immune reactions betweenlymphocytes and bodies recognized as foreign.

As used herein, such bodies can include any living or dead biologicalmaterial that is delivered to or invasive to the body of a mammal,including a human. Antigenic such bodies are those which in the normalcourse elicit an immune response either by the recipient or by the body,for instance where the body itself comprises immune cells includinglymphocytes, such as a bone marrow, tissue or organ graft. Alloantigenicbodies are bodies that are antigenic between individuals within the samespecies; xenoantigenic bodies are antigenic between individuals ofdifferent species. Autologous bodies are bodies from the recipient. Inembodiments, the bodies are HLA mismatched bodies. In other embodiments,the bodies comprise HLA mismatched lymphocytes.

While the mechanism of HUCPVC immunomodulatory action is not completelyunderstood, it is expected that the HUCPVCs and soluble factors producedby them have an effect on the major cell populations involved inalloantigen recognition and elimination, such as antigen presentingcells, T cells including cytotoxic T cells, and natural killer cells.

The HUCPVCs useful in the present method are described in theliterature, as noted hereinabove, and are characterized moreparticularly as progenitor cells extractable from the perivascularregion of umbilical cord, including but not limited to human umbilicalcord. Using the protocol described herein, it will be appreciated thatumbilical cord perivascular cells can also be extracted from theumbilical cord vasculature of other mammals, including horses, cows,pigs, primates and the like. The perivascular region comprises theWharton's jelly associated with and external of the umbilical cordvasculature. The HUCPVCs are extractable from the Wharton's jelly thatlies in the perivascular region, using standard methods of digestionsuch as with collagenase or related enzymes suitable for removingassociated connective tissue, as described for instance by Sarugaser etal, 2005, the entire contents of which are incorporated herein byreference. Preferably, HUCPVCs are harvested only from the perivascularcells, and not from Wharton's jelly extending beyond the perivascularregion, or from tissues or fluids that are part of or internal to thevasculature itself. This avoids contamination by other cells within thecord generally. In the alternative, extraction from the Wharton's jellywithout selection for perivascular cells can be performed, provided theresulting cell population is enriched for HUCPVCs using for instanceflow cytometry to enrich for progenitor cells having the phenotype andcharacteristics noted herein. The HUCPVCs further are characterized byrelatively rapid proliferation, exhibiting a doubling time, in each ofpassages 2-7, of about 20 hours (serum dependent) when cultured understandard adherent conditions. Phenotypically, the HUCPVCs arecharacterized, at harvest, as Oct 4-, CD14-, CD19-, CD34-, CD44+, CD45-,CD49e+, CD90+, CD105(SH2)+, CD73(SH3)+, CD79b-, HLA-G-, CXCR4+, c-kit+.In addition HUCPVCs contain cells which are positive for CK8, CK18,CK19, PD-L2, CD 146 and 3G5 (a pericyte marker), at levels higher thanin cell populations extracted from Wharton's jelly sources other thanthe perivascular region.

HUCPVCs can also express variable levels of MHC class I, from 0-100%dependent upon manipulation. By subjecting harvested cells to afreeze-thaw cycle, as described for instance by Sarugaser et ah, 2005,incorporated herein by reference, one obtains a HUCPVC population thatis substantially negative for both MHC class I and MHC class II (95%).As used herein, the HUCPVCs are considered to be “substantially”negative for MHC class I and MHC class II if the number of cellsresident in a given population and expressing either one or bothphenotypes is not more than about 20% of the cell population, e.g., notmore than 15%, 10%, 5% or less of the total HUCPVC population. Adetermination can be made using standard techniques of flow cytometrywith appropriate tagged antibody. This MHC double negative HUCPVCpopulation is particularly useful in the methods of the presentinvention. It will be appreciated that, in the present method, theadministered HUCPVC population can comprise either freshly extracted(optionally expanded) MHC class I negative cells, or the thawed MHCdouble negative HUCPVCs. The MHC double negative HUCPVCs are far lesslikely to stimulate an immune response in a recipient, and theirclinical use is accordingly preferred herein. It should be appreciated,however, that manipulation of the MHC phenotype is not essential. Theimmunoprivilege and immunomodulation properties are seen also in freshlyextracted HUCPVCs that have optionally been stored, and not only inHUCPVCs that have been manipulated by freeze/thaw.

In the present method, HUCPVCs are exploited for their immunoprivilegedand immunomodulatory properties, in clinical setting that would benefittherefrom. The term “immunoprivileged” is used herein with reference tocells, such as HUCPVCs, that when incubated with peripheral bloodlymphocytes, either do not stimulate PBL proliferation to astatistically significant extent, or retain their viability at astatistically significant level, particularly when tested using theso-called one-way MLC assay established in this art and exemplifiedherein.

The term “immunomodulatory” is used herein with reference to the abilityof HUCPVCs to mitigate, reduce or inhibit the reaction betweenmismatched populations of lymphocytes, as revealed either by a reductionin the mortality of a lymphocyte population, or by an increase in theviability of a lymphocyte population, as determined using, for instance,the so-called one- or two-way mixed lymphocyte reaction (MLR).

It has in addition been found that factors exuded by cultured HUCPVCscan alone exert an immunomodulatory effect, of the type seen when intactHUCPVCs are used. Thus, in aspects and embodiments of the presentinvention, the extracted soluble factors produced upon culturing ofHUCPVCs are used either alone or in combination with the HUCPV cells, asimmunomodulators.

The one or more immunomodulatory factors exuded upon HUCPVC culturingare referred to herein a soluble factors, and are extractable from themedium in which HUCPVCs are cultured. In one embodiment, theimmunomodulating soluble factors are provided as an extract obtainedwhen HUCPVC cells are removed from the medium conditioned by theirgrowth, such as by centrifugation. When centrifugation is employed, theextract is provided as the supernatant. Suitable HUCPVC culturingconditions are exemplified herein. The extract is obtained by separatingthe cells from the conditioned culturing medium, such as bycentrifugation. In other embodiments, the soluble factors are providedas an immunomodulating fraction of such extract. An extract fractionhaving immunomodulating activity is also useful herein, and can beidentified using the mixed lymphocyte reactions described herein. Theseextract fractions can of course be obtained by fractionating the HUCPVCextract using any convenient technique including solvent extraction,HPLC fractionation, centrifugation, size exclusion, salt or osmoticgradient fractionation and the like. Eluted or collected fractions canthen each be subjected to the MLR and fractions active forimmunomodulation can be identified, and a fraction with immunomodulatingactivity can be used clinically in the manner described herein.

Thus, in embodiments, the present invention comprises the use ofimmunomodulating extracts or immunomodulating fractions thereofcomprising soluble factors exuded during culturing of HUCPVCs.

Use of the HUCPVCs, and populations thereof that are immunoprivilegedand/or immunomodulatory, in accordance with the present invention,entails their collection, optionally their expansion, further optionallytheir cryogenic storage and revival from the frozen state, and theirsubsequent formulation for administration to the intended recipient. Theparticular manipulation, dosing and treatment regimen will of coursedepend on numerous factors, including the type and severity of thedisease or condition to be treated. For immunological conditions (eg.GvHD, autoimmune conditions), the size of the HUCPVC population, i.e.,the dose administered to the recipient, will lie generally in the rangefrom 0.01 to about 5 million cells per kilogram of recipient bodyweight. For delivery, the cells are provided suitably as a formulationfurther comprising a physiologically tolerable vehicle, i.e., a vehiclethat is tolerable not only by the cells but also by the recipient.Suitably, the cells are provided in a sterile formulation comprising, ascarrier, a physiologically tolerable vehicle such as saline, bufferedsaline such as PBS, cell culture medium or similar liquid containing anyof: essential amino acids, growth factors, cytokines, vitamins,antibiotics or serum-free chemically defined media etc, or sterilewater. The formulated cells can be administered by infusion, or byinjection using for instance volumes in the 1-25 mL range.

The immunomodulating soluble factors produced by HUCPVC and extractablefrom spent HUCPVC culturing medium are similarly useful in the mannerdescribed above with reference to intact HUCPVCs. In one embodiment, theextract itself constitutes the pharmaceutical composition, thuscomprising the active agent in the form of immunomodulating solublefactor, and the medium constituting the physiologically tolerablevehicle. In the alternative, the extract can be dried, to retain thesoluble factor(s) and reconstituted in a different vehicle, such asphosphate buffered saline. The dosage size and dosing regimen suitablefor clinical applications can be determined with reference to the dosingeffective for intact HUCPVCs. The dose equivalent of extract can bedetermined by calculating the relative potencies of the extract andintact cells in the MLR assay, or any alternative thereto which isreflective of the clinical environment in which the therapy will beexploited, such as in appropriate animal models of the targetedindication.

In use, the formulated HUCPVCs or soluble factors produced thereby areadministered to treat subjects experiencing or at risk of developing anadverse immune reaction. Such subjects include particularly subjectsreceiving or about to receive an allogeneic or xenogenic transplant orgraft, in the form of cells such as marrow and peripheral blood, tissuesincluding skin and vascular tissue including coronary tissue andgastrointestinal tissue, or an organ such as liver, kidney, heart, lung,etc. The formulated HUCPVCs are useful particularly to reduce the onsetor severity of graft versus host disease, a condition resulting from animmune attack of host tissues mediated by lymphocytes present in thedonor graft. In one embodiment of the invention, the HUCPVCs can be usedto treat the graft by incubation therewith for a period of time, priorto transplantation, sufficient to reduce or arrest the activity oflymphocytes resident in the graft. This incubation would require HUCPVCs(from either fresh or frozen stock) to be included at a dose of 5-60% oftotal graft lymphocytes (as determined by the volume of blood present inthe graft), preferably 10-40% for between 4 and 10 days, in order tohalt proliferation prior to implantation. In the case of an organ graft,the organ would be incubated with HUCPVCs (suspended in aphysiologically tolerable vehicle as mentioned above), from either freshor frozen stock, at a dose of 0.01 to 5×10³ cells per gram mass of theorgan, prior to implantation, for a period of time to cause the organ'slymphocytes to become inactive. For subjects that are graft recipients,the HUCPVCs desirably are administered by in the tissue directlysurrounding the allogeneic organ to the recipient prior to (e.g. withinhours of), concurrently with, or after grafting (e.g., within hoursafter, and thereafter as necessary to control immune reaction). TheHUCPVCs can be administered, most suitably at the site of the graft,such as by infusion or injection, either subcutaneously,intramuscularly, intravasculary, intravenously, intraarterially, orintraperitoneally. In one embodiment, the recipient is treated at thetime of grafting by infusion with HUCPVC doses in the range from 5×10⁴to 5×10⁷ cells per kg body weight, such as about 1 to 5×10⁶ cells. Thecells are formulated in 10 ml of normal saline with 5% human serumalbumin. Two or more infusions can be used, each lasting about 10-15minutes. The cells can also be implanted in a slow release formulationthat allows the release of viable cells over time at the implantationsite (such as intraperitoneal, intramuscular etc). Carriers suitable forthis purpose include gelatin, hyaluronic acid, alginate and the like. Inanother embodiment of the invention, HUCPVCs can be utilized to treatimmunological conditions such as GvHD which are underway, and possiblyrefractory to other treatments. The HUCPVCs or the exuded solublefactors thus will be useful to treat subjects afflicted with leukemias,aplastic anemias and enzyme or immune deficiencies for whom thetransplantation of immune cells or tissues containing them areindicated.

The administration of HUCPVCs or the soluble factors also hasapplication in treating autoimmune diseases such as Crohn's disease,lupus and multiple sclerosis, as well as rheumatoid arthritis, type-1insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopernic purpura, Sjogren's syndrome, encephalitis, uveitis,leukocyte adhesion deficiency, rheumatic fever, Reiter's syndrome,psoriatic arthritis, progressive systemic sclerosis, primary biliarycirrhosis, myasthenia gravis, lupus erythematosus, vasculitis,pernicious anemia, antigen-antibody complex mediated diseases, Reynard'ssyndrome, glomerulonephritis, chronic active hepatitis, celiac disease,autoimmune complications of AIDS, ankylosing spondylitis and Addison'sdisease. The administration of HUCPVCs in this case is intravenously ina physiologically tolerable vehicle (as mentioned previously), with adose ranging from 0.1-10×10⁶ cells/kg body weight. More than one dosemay be required, and dosing can be repeated as needed.

Secondly, HUCPVCs can be used to treat protein/enzyme deficiencies,wherein the HUCPVCs have been transfected with the gene necessary toproduce the desired protein/enzyme. This process can includetransduction (including but not limited to: lentiviral, retroviral andadenoviral); and transfection (including but not limited to:nucleofection, electroportation, liposomal) and are transfused into apatient suffering from a deficiency. The dose administered to therecipient will lie generally in the range from 0.01 to about 5 millioncells per kilogram of recipient body weight. HUCPVCs will then producethe protein or enzyme of interest constitutively

Finally, HUCPVCs can be engineered to introduce transgenes, via thetransfection methods mentioned above, for use as vaccines in order togenerate large quantities for administration to people at risk ofexposure to specific viruses/antigens.

MATERIALS AND METHODS

Cell Harvests

HUCPVCs

Ethical consent for this research was obtained from the University ofToronto as well as Sunnybrook & Women's College Health Sciences Centre.Umbilical cords were collected from aseptic caesarean births of fullterm babies, upon obtaining informed consent from both parents. Thecords were immediately transported to the University of Toronto wherecells were extracted from the perivascular area under sterile conditionsas reported previously¹⁶. Briefly, 4 cm sections of cord were cut, andthe epithelium was removed. The vessels were then extracted includingtheir surrounding Wharton's jelly, tied in a loop to prevent smoothmuscle digestion, and digested overnight in a collagenase solution. Uponremoval from the digest the following day, the cells were rinsed inammonium chloride to lyse any red blood cells from the cord blood.Following this, the cells were rinsed and plated out in 85% α-MEMcontaining 5% fetal bovine serum and 10% antibiotics (penicillin G at167 units/ml; Sigma, gentamicin 50 μg/ml; Sigma, and amphotericin B 0.3μg/ml) at a density of 4,000 cells/cm². The cells are passaged when theyreach 75-80% confluence, which is approximately every 6-7 days.

MHC−/− HUCPVCs

Test cell populations of >1×10⁵ cells were washed in PBS containing 2%FBS and re-suspended in PBS+2% FBS with saturating concentrations (1:100dilution) of the following conjugated mouse IgG1 HLA-A,B,C-PE (BDBiosciences #555553, Lot M076246) (MHC I), HLA-DR,DP,DQ-FITC (BDBiosciences #555558, Lot M074842) (MHC II) and CD45-Cy-Cychrome (BDBiosciences #555484, Lot 0000035746) for 30 minutes at 4° C. The cellsuspension was washed twice with PBS+2% FBS and re-suspended in PBS+2%FBS for analysis on a flow cytometer (XL, Beckman-Coulter, Miami, Fla.)using the ExpoADCXL4 software (Beckman-Coulter). Positive staining wasdefined as the emission of a fluorescence signal that exceeded levelsobtained by >99% of cells from the control population stained withmatched isotype antibodies (FITC-, PE-, and Cy-cychrome-conjugated mouseIgG1, κ monoclonal isotype standards, BD Biosciences), which wasconfirmed by positive fluorescence of human BM samples. For each sample,at least 10,000 list mode events were collected. All plots weregenerated in EXPO 32 ADC Analysis software.

The attached cells were sub-cultured (passaged) using 0.1% trypsinsolution after 7 days, at which point they exhibited 80-90% confluencyas observed by light microscopy. Upon passage, the cells were observedby flow cytometry for expression of MHC-A,B,C, MHC-DR,DP,DQ, and CD45.They were then plated in T-75 tissue culture polystyrene flasks at 4×10³cells/cm² in SM, and treated with 10⁻⁸ M Dex, 5 mM β-GP and 50 μg/mlascorbic acid to test the osteogenic capacity of these cells. Theseflasks were observed on days 2, 3, 4, 5 and 6 of culture for CFU-O, orbone nodule, formation. Any residual cells from the passaging procedurealso were cryopreserved for future use.

Aliquots of 1×10⁶ PVT cells were prepared in 1 ml total volumeconsisting of 90% FBS, 10% dimethyl sulphoxide (DMSO) (Sigma D-2650,Lot#11K2320), and pipetted into 1 ml polypropylene cryo-vials. The vialswere placed into a −70° C. freezer overnight, and transferred thefollowing day to a −150° C. freezer for long-term storage. After oneweek of cryo-preservation, the PVT cells were thawed and observed byflow cytometry for expression of MHC-A,B,C, MHC-DR,DP,DQ, and CD45. Asecond protocol was used in which the PVT cells were thawed after oneweek of cryopreservation, recultured for one week, sub-cultured thenreanalyzed by flow cytometry for expression of MHC-A,B,C, MHC-DR,DP,DQ,and CD45.

It was noted that the frequency of MHC−/− within the fresh cellpopulation is maintained through several passages. When fresh cells arefrozen after passaging, at −150° C. for one week and then immediatelyanalyzed for MHC phenotype, this analyzed population displays aremarkably enhanced frequency of cells of the MHC−/− phenotype. Inparticular, first passage of cryopreserved cells increases the relativepopulation of MHC−/− cells to greater than 50% and subsequent freezingand passaging of those cells yields an MHC−/− population of greater than80%, 85%, 90% and 95%.

Lymphocytes

Peripheral blood lymphocytes (PBLs) were extracted from heparinizedblood from healthy donors. Cell separation was achieved by Ficoll-Paque™PLUS density gradient (Amersham Biosciences #17-1440-03) in which thecells were spun for 35 minutes at 380×g. The buffy coat was removed andcounted using a ViCell-XR™ (Beckman Coulter) with a protocol specificfor lymphocytes as determined by cell and nucleus size. The cells werethen plated out as per the requirements of the assay in 80% RPMI-1640media (Sigma #R5886) containing HEPES (25 mmol/L), L-Glutamine (2mmol/L), 10% fetal bovine serum and 10% antibiotics.

Mixed Lymphocyte Cultures

Mitomycin C

In order for one-way MLCs to be performed, the HUCPVCs and one of thePBL populations have to remain quiescent. This is achieved by treatingthe cells with mitomycin C (MMC) at a set concentration and time,allowing the MMC to adhere to the DNA and prevent division. Thisconcentration was determined by a titration curve of MMC incubated for20 minutes at 37° C. (5% CO₂) with a starting cell population of 5000cells in a well of a 96 well plate. Concentrations of 10, 20, 30, 40,50, and 75 μg/mL of MMC were tested in regular media (85% α-MEM, 5% FBS,10% antibiotics) and washed twice with PBS afterwards to remove anytraces of the MMC. The wells were counted after a week to assessproliferation. It is essential that all traces of MMC are removed so itdoes not affect the proliferative capacity of lymphocytes when the cellpopulations are combined in an MLC. To ensure this, empty wells of a 96well plate (Falcon) were treated with MMC and washed as per theprotocol. Lymphocytes were then added and assayed for theirproliferation compared to control normal wells.

Iminunoprivilege Assays

Triplicates of 1×10⁴ HUCPVCs (both fresh and frozen have been assayed)were plated in 96 well plates (n=5). Once the cells had attached (afterapproximately 2 hours), they were treated with MMC at 20 μg/mL. TheHUCPVCs were then rinsed, and 10⁵ PBLs from Donor 1 were added to eachwell. The plates were incubated at 37° C. with 5% CO₂ air in 80%RPMI-1640 media containing HEPES (25 mmol/L), L-Glutamine (2 mmol/L),10% fetal bovine serum and 10% antibiotics. After 6 days the lymphocytespresent in culture with HUCPVCs were counted using the ViCell counter,and compared to controls. For the cell death assay, plates were allowedto incubate for four hours, and HUCPVCs were assessed for early and latestage cell death markers; annexin 5 (R&D Systems TA4638) and7-amino-actinomycin D (7-AAD) respectively. These levels were measuredand compared using Flow Cytometry on a Beckman Coulter FlowCenter. Forthe PBL proliferation assay, the cells were allowed to incubate for 6days, after which they were stained with 5-bromo-2-deoxyuridine (BrdU),a base analog of thymidine, and measured using flow cytometry. Controlsof PBLs alone and HUCPVCs alone were used for both assays.

For one-way MLCs, HUCPVCs were first plated in a 96 well plate intriplicate (1, 2, 3, and 4×10⁴ cells per well), treated with MMC andwashed with PBS. PBLs were retrieved from two unmatched donors selectedfrom a pool of potential donors (Mismatch on 5 out of 6 HLA tested:Donor 1 HLA-A*01, *02; B*07, *18; DRB1*15, *—; Donor 2 A *01, *—; B*08,*—; DRB1 *03, *—). Typing was performed at the RegionalHistocompatibility Laboratory (Toronto General Hospital, Toronto, ON)using DNA assignment techniques at low resolution. After Ficolling,Donor 2's lymphocytes were treated with MMC to be quiescent. The cellswere then spun down and washed, and added to a 96 well plate at 10⁵cells per well. Donor 1's lymphocytes were added at 10⁵ cells per welland the three cell populations were allowed to co-incubate for 6 days,after which they were stained with 5-bromo-2-deoxyuridine (BrdU), a baseanalog of thymidine. Flow cytometry for BrdU was then performed on thelymphocytes to assay proliferation. Following this, a similar assay wasperformed with the result being measured using daily counts oflymphocytes (from day 1 to 6) using the ViCell-XR™ cell counter, with alymphocyte-specific protocol. All results were compared to allogeneicand autologous controls from both donors.

Immunomodulation Assays

Two-way MLCs incorporate two PBL populations from unmatched donors (samedonors as above), both of which are permitted to proliferate. Briefly,1×10⁵ PBLs from both donors were added to each well of a 96 well plateand left to incubate for six days. In the course of the experiment,either 1 or 4×10⁴ HUCPVCs were added to wells in triplicate on days 0,three or five in order to analyze the effectiveness of HUCPVCs if animmune reaction has already begun. The 10 and 40% HUCPVC:PBL ratios werechosen as both had showed positive results previously, and thus werechosen as the low and high levels of HUCPVC inclusion. Samples from eachplate were counted every day using the ViCell-XR™ cell counter andcompared to autologous and allogeneic controls.

Soluble Factor

The two-way MLC assay was performed again, without direct HUCPVC to PBLcell contact to determine if the effect noted was due to a solublefactor, or if cell-cell contact was necessary. The HUCPVCs (1 and 4×10⁴cells per well) were cultured on a Transwell® insert (Corning) for a 24well plate, and allowed to attach for approximately 2 hours. Once theyhad attached, the insert was transferred to the 24 well plate thatcontained a co-culture of PBL populations from Donor 1/Donor 2 (samedonors as above) (n=3). The lymphocyte cell numbers were counted dailyfor six days and compared to autologous and allogeneic controls usingthe ViCell-XR™.

Lymphocyte Activation

Both immunoprivilege assays and two-way MLCs were performed as mentionedpreviously. The endpoint of this assay was flow cytometric analysis ofthe lymphocytes for the presence of IL-2R (CD25) (Becton Dickinson,#555431), a marker of activated lymphocytes. This assay was performedover 6 days to determine if HUCPVCs caused an increase or decrease inlymphocyte activation. Lymphocytes were also co-stained with CD45 toensure proper cell population was obtained. Negative controls werelymphocyte cultures with no HUCPVCs added, and unstained.

Activated T Cell Line Generation

PBLs were extracted from Donor 1 and 2 as previously, and separatedusing the Ficoll gradient. The cells were counted, and cells from Donor2 were rendered quiescent with MMC. 10⁶ Cells from Donor 1 were platedin a 24 well plate, and stimulated with a 1:1 ratio of quiescent PBLsfrom Donor 2. The cells were fed with 2 mLs of RPMI-1640 media(supplemented with 10% serum and 10% supplements as previously) andallowed to activate. Media was changed upon a perceptible change of itscolour to yellow (˜3 days). After ˜11 days (or when the media changedcolour in under 3 days), the cells were harvested and counted. They werere-plated at 10⁶ per well, and re-stimulated with a 1:1 ratio ofquiescent PBLs from Donor 2. Upon the second stimulation, IL-2 was addedat a concentration of 100 U/mL (BD Biosciences #354043), every 2 dayswith feeding. When media turned yellow before 3 days, the cells wereharvested, counted and split. Following this procedure, the cells wereready to be used as Activated T lymphocytes (ATLs), with specificantibodies to Donor 2. The PBL co-culture and two-way MLCs were carriedout as previously and assayed for cell proliferation and expression ofCD25.

Lymphocyte Labelling

In order to visualize, and quantify, the difference between twolymphocyte populations, PKH26 was used (Sigma #PKH26-GL). PHK26 is anon-cytotoxic membrane dye with a long half life (˜100 days). Cells werestained as per the protocol supplied with the product: lymphocytes weretrypsinized, pooled, and pelleted; the diluted dye was then added to thecell suspension for 2 to 5 minutes (2 mL of 2×10⁻⁶ molar PHK26solution). After staining, an equal volume of serum was added to haltthe reaction; the cells were suspended in media, spun down and washedseveral times. The stain was then visualized on the fluorescencemicroscope to ensure appropriate dye uptake resulted. The cells used forstaining were the ATLs obtained from Donor 1; these red cells wereincluded in an MLC with unstained cells from Donor 2, and HUCPVCs. Theendpoint of the assay was flow cytometry for CD45 (BD #555482) and CD25,gated on the presence or absence of PKH26. Negative controls wereunstained cells, and MLCs with no HUCPVCs

Transfection

First, 293 Cells are transfected with the desired DNA and plasmids(vector DNA, 10 μg gag/pol expressing plasmid, 10 μg of rev expressingplasmid, 10 μg of tat expressing plasmid, 5 μg of VSV-G expressingplasmid with 2.5 M CaCl₂). These are allowed to incubate overnight,after which the media is changed. Cells are then left with this mediafor three more days, after which the supernatant of the cells iscollected and filtered. The viral supernatant is then concentrated withultracentrifugation (50000 g for 90 minutes) or using an Amicon Ultra-15Centrifugal Filter device (100,000 MWCO; Millipore). When this processis complete, the viral supernatant can be combined with the HUCPVCs at aconcentration determined by titering the concentrated virus, and allowedto incubate overnight. The following day, more media is added, and thecells incubate for 6 more hours before changing the media. In thismanner, HUCPVCs can be engineered to introduce and express a transgenethat encodes any protein, including proteins useful to manage adverseimmune reactions (immunosuppressive proteins). Such proteins includeCTLA4, VCP, PLIF, LSF-1, Nip, CD200 and Uromodulin.

Microarray Analysis

The Oligo GEArray Human Cancer Microarray (Superarray Biosciences,Frederick Md., Cat#: OHS-802) was used to find changes in the expressionof genes representative of several different pathways frequently alteredduring the progression of cancer The Oligo GEArray for human cancer has440 representative cancer genes and is organized into functional genegroupings including apoptosis, cell cycle, cell growth anddifferentiation, signal transduction, and other cancer-related genes.

HUCPVCs and human bone marrow-derived MSCs were grown to passage 2 andRNA was isolated from these cells. Purified RNA was processed accordingto manufacturer's protocol (Superarray Biosciences Corp.) and hybridizedto Oligo GEArray Human Cancer microarrays. The Oligo GEArray HumanCancer Microarray was used to determine the differential expression ofgenes related to cancer in HUCPVCs compared with normal human bonemarrow-derived MSCs.

RESULTS

Mitomycin C is an Effective Antiproliferative Agent on HUCPVCs

HUCPVCs were treated with a range of concentrations of MMC for 20minutes at 37° C. (5% CO₂). FIG. 1 shows the cell numbers of HUCPVCsafter one week in culture post-MMC treatment (n=2). All cells show amarked decrease in proliferation relative to control (p<0.001), with nodifference among treatments. Thus, 20 μg/mL was chosen in accordancewith the literature. FIG. 2 illustrates the lack of effect of cellsplated in wells treated with MMC and washed, versus untreated wells(n=2, p=0.16). Therefore, the MMC will not have an effect onexperimental results obtained in wells previously treated with MMC. Allstatistics presented herein were obtained using ANOVAs to compare meansvia the R Project for Statistical Computing.

HUCPVCs are not Recognized as Foreign by Lymphocytes

Upon co-incubation of a HUCPVC population with lymphocytes (Donor 1),there was a statistically significant increase in HUCPVC death at aproportion of 10% HUCPVC:PBL (n=5, p=0.01), as measured by annexin 5mean fluorescence intensity (MFI). FIG. 3 shows this increased celldeath was not noted at HUCPVC doses higher than 10%, thus at the correctproportion, HUCPVCs are not attacked by unmatched lymphocytes. This wasconfirmed using a lymphocyte proliferation assay in which it can be seenthat lymphocytes do proliferate in response to 10% HUCPVC:PBL, asmeasured by BrdU MFI (p=0.02), but not at higher HUCPVC concentrations(FIG. 4). Lymphocyte proliferation is a standard measure of activation,as division of both T and B lymphocytes occurs in the activationcascade. Therefore at a lower proportion, HUCPVCs do not provide enoughof a presence such that their immunological avoidance capabilities arerealized. However at higher concentrations, 20-40%, the PBLs do notproliferate, and the HUCPVCs are not killed.

HUCPVCs were also analyzed for their effect on lymphocyte cell numberupon inclusion in a co-culture with resting PBLs or ATLs. In both cases,HUCPVCs caused no significant increase over control cell number (PBL:35.2±3.1×10³, +10% HUCPVCs 45.0±5.7×10³; ATL: 38.8±18.2×10³, +10%HUCPVCs 40.8±4.8×10³), indicating their immunoprivilege in eitherresting or stimulated conditions (FIG. 7).

HUCPVCs were included in a one-way MLC in proportions of 10, 20, 30 and40% of the PBL population and assessed for their proliferation by BrdUexpression after 6 days. FIG. 8 shows no significant increase in thenumber of cells proliferating regardless of the proportion of HUCPVCsincluded.

HUCPVCs Are Immunomodulatory

FIG. 5 shows that on day 6 the allogeneic co-cultured lymphocytes haveincreased in number, whereas all cultures with HUCPVCs present;regardless of when they were added or the proportion added, have asignificantly lower lymphocyte cell count than the control (n=3).

HUCPVCs Can Exert Their Action Through A Soluble Factor

Trans Well® inserts were used to separate HUCPVCs from PBLs in a two-wayMLC. No significant reduction in lymphocyte number relative to controlwas seen within any day with either 10% or 40% HUCPVCs (n=3) (FIG. 6).However, upon increasing the sample number, the addition of 10% HUCPVCsshowed a significant reduction in lymphocyte cell number over a 6 dayculture period compared to control (MLC: 40.7+32.9×10³ cells, 10%HUCPVCs: 21.3±14.7×10³ cells) (FIG. 9). Soluble factor(s) may thereforecontribute to HUCPVC immunomodulation, however what that factor(s)is/are and how they affect lymphocytes is still unknown.

HUCPVCs Reduce the Activation State of Lymphocytes

ATLs stained with PKH26 were added in a co-culture with 10% HUCPVCs, andassayed for their expression of CD25 (IL-2 receptor), a marker oflymphocyte activation. Upon inclusion of HUCPVCs, both the percentage ofcells expressing CD25 (Control: 100%±0, 10% HUCPVC: 96.9%±0.7), and themean fluorescence intensity (Control: 28.6±0.1, 10% HUCPVC: 3.76±0.1)was significantly reduced (FIG. 10). Thus, HUCPVCs have a physicaleffect on activated lymphocytes, by reducing their activation state.

In addition, HUCPVCs reduced the expression of CD45 of the lymphocytes,both the percentage (Control: 100%±0, 10% HUCPVC: 99.6%±0.1, 40% HUCPVC:98.4%±0.36) and the mean fluorescence intensity (Control: 28.20±4.24,10% HUCPVC: 16.33±1.27, 40% HUCPVC: 14.70±1.22) were significantlydifferent (p<0.05) (FIG. 11). These results were unexpected as CD45 isexpressed on all lymphocytes. However it has been seen that CD45 iscrucial for the development and function of lymphocytes³⁶, and may be afurther indication of the reduced function of the lymphocytes due to theaddition of the HUCPVCs.

Transfection

HUCPVCs were successfully transfected with GFP, and the cells expressedhigh levels of the protein. A transfection efficiency of 97% wasachieved (FIG. 12), with maintenance of good proliferative rates. Thissuccess rate varies according to the passage at which the cells aretransfected. With a functioning transfection protocol, it is thuspossible to transfect the cells with any protein and have itconstitutively expressed.

Microarray Analysis

HUCPVCs do not express any detectable levels of genes associated withtumorigenesis. The gene array analysis resulted in the absence offunctional genes associated with human cancer and expressed genes knownto be important, for example, in cell cycle regulation, such as CyclinD1 (CCND1), CCND2 and CCND3 (FIG. 13).

DISCUSSION

Herein described are the in vitro immunoprivileged and immunomodulatoryproperties of an MSC population from a source other than bone marrow,the human umbilical cord. HUCPVCs are extracted from the perivasculararea of the cord, as this was believed to be the most rapidlyproliferating population of cells. Previously, it was shown thatendothelial cells from the wall of the umbilical cord vein stimulatedlymphocytes in vitro³⁷. This is in stark contrast to the resultsreported, and reinforces the distinct area from which HUCPVCs areretrieved.

HUCPVCs thus are well suited for clinical use particularly, but notonly, to reduce the onset and/or severity of graft versus host disease,and to reduce or eliminate graft rejection by the host, and for thetreatment of other immune-mediated disorders that would benefit fromsuppression of a mixed lymphocyte reaction. In addition, whenmanipulated by transfection to contain the gene of a protein/enzyme ofinterest, HUCPVCs are able to produce this product constitutively, andwould thus be useful in the treatment of any condition in which aprotein/enzyme deficiency results in a detrimental effect to thepatient, especially as they can be used allogeneically in a mismatchedpatient without rejection. In addition, HUCPVCs can also be used togenerate vaccines of interest after transfection with the necessarygene.

As progenitor cells having the propensity to expand and differentiateover time into various mesenchymal tissues dictated by their growthenvironment, HUCPVCs like other mesenchymal progenitor or stem cells maycarry some risk that their growth and differentiation in vivo will notbe controlled. Remarkably, as a further benefit of the use of HUCPVCsclinically, it has been determined that the HUCPVCs exhibit extremelylow telomerase activity, an indicator of their propensity fortumorigenesis. Furthermore, it has been determined that HUCPVCs lackmany of the genetic markers that are hallmarks of tumorigenesis.Tumorigenesis occurs by mutations that deregulate biological pathwaysand cause cells to grow and divide unchecked, to avoid apoptosis(programmed cell death), to respond abnormally to growth factors, toreceive blood supply (angiogenesis), and to migrate from one location toanother (metastasis and invasiveness). Many genes are involved in eachof these control mechanisms, and a mutation in any one of them can causederegulation.

The following references are incorporated hereby by reference in theirentirety:

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We claim:
 1. An in vivo method for treating a subject having or at riskof developing an adverse immune reaction, comprising administering tothe subject a composition comprising an immunomodulating effectiveamount of the soluble fraction of medium conditioned by umbilical cordperivascular cells, wherein said adverse immune reaction is directedagainst biological material other than said composition and wherein saidadministering treats said subject by inhibiting or reducing said adverseimmune reaction.
 2. The method of claim 1, wherein the subject has or isat risk for graft versus host disease.
 3. The method of claim 1, whereinthe subject has or is at risk for a mixed lymphocyte reaction.
 4. Themethod of claim 1, wherein the subject has or is at risk for graftrejection.
 5. The method of claim 4, wherein the graft is a skin graft.6. The method of claim 4, wherein the graft is an organ graft.
 7. Themethod of claim 4, wherein the graft is a marrow graft.
 8. The method ofclaim 4, wherein the graft is a peripheral blood graft.
 9. The method ofclaim 1, wherein the subject has an autoimmune disorder.
 10. The methodof claim 1, wherein the subject is afflicted with a leukemia and is atrisk for graft versus host disease.
 11. The method of claim 1, whereinthe umbilical cord perivascular cells are human umbilical cordperivascular cells (HUCPVCs).
 12. The method of claim 1, wherein theumbilical cord perivascular cells comprise a transgene that encodes aprotein of interest.
 13. The method of claim 11, wherein the umbilicalcord perivascular cells are substantially MHC double negative HUCPVCs.14. The method of claim 1, wherein the second component comprises anunfractionated extract of medium conditioned by said cells thatcomprises said soluble fraction.
 15. The method of claim 1, wherein thecomposition further comprises said umbilical cord perivascular cells.16. The method of claim 15, wherein said umbilical cord perivascularcells are human umbilical cord perivascular cells (HUCPVCs).